Extended Data Fig. 6: The G3P shuttle is not involved in the establishment of senescence.
A: Nodes with the highest betweenness values (top 20) in the overlap network produced by intersecting a Myoblast-only Gene–Metabolite Correlation Network, which considers genes and metabolites presenting a correlation with absolute value higher than 0.5 in myoblast samples, and the Fibroblast Gene-Metabolite Correlation Network, considering all senescence inducers (RAS- and RAF-OIS, DDIS, and RS) and quiescence (Q). B: Genes (green squares) whose expression correlates either positively or negatively with G3P accumulation during senescence in WI38 fibroblasts. The size of green squares is proportional to node betweenness for each target. C: Representative Western blots showing indicated protein levels in extracts of WI38 fibroblasts proliferating or undergoing DDIS for 7 days. D: Activity of mitochondrial Glycerol-3-phosphate dehydrogenase calculated from the measurement of glycerol-3-phosphate cytochrome c reductase in WI38 fibroblasts undergoing DDIS or RAS-OIS during 7 days. n = 2 biological replicates. E: Representative Western blots showing indicated protein levels in WI38 fibroblasts proliferating (Prolif.) or undergoing RAS-OIS induction and not infected (-) or infected with an adenovirus overexpressing GFP or GPD1 for 7 days. F: Densitometric quantification of p16 and p21 protein levels relative to actin from three experiments, including the one of the panel, in proliferative, RAS-OIS cells infected with GFP- or GPD1-overexpressing adenoviruses for 7 days. G: mRNA levels scored by RT–qPCR in WI38 fibroblasts proliferating (Prolif.) or undergoing RAS-OIS and infected with an adenovirus carrying a control scramble shRNA (shCtrl) or an shRNA targeting GPD1 (shGPD1) for 7 days. H: Maximal fold change of the GK mRNA in WI38 fibroblasts and myoblasts induced to senesce under the indicated conditions relative to proliferating cells as measured by Affymetrix microarrays (n = 2 for all the samples, except n = 4 for the senescence sample of RS and n = 3 for proliferative samples of DDIS fibroblasts and RAS-OIS myoblasts). Data are presented as mean values. I: Measurement of glycerol uptake in WI38 fibroblast proliferating, undergoing RAS-OIS (7 days) or DDIS (10 days). The reported values are relative to those of proliferating cells. For panels F, G, E, bars represent the means of 3 biological replicates +/- s.d. Indicated p values were calculated using an unpaired two-sided Student’s t-test.
